Nitroderivatives of polysaccharides

ABSTRACT

The present invention provides a new class of compounds presenting a high compatibility with tissues and organic fluids. Such new compounds are polysaccharides essentially formed of units of uronic acid and/or hexosamine, containing nitro groups —ONO 2  covalently bonded to the saccharide structure. Preferably, the polysaccharides according to the invention are prevalently formed of disaccharide repeating units formed of uronic acid and hexosamine. These compounds, in psychological conditions, selectively release NO, allowing a reduction in the amount of NO needed to achieve a determined therapeutical effect. This result has been achieved by functionalizing polysaccharides essentially formed of units of uronic acid and/or hexosamine, with subsituents containing a ONO 2 ? group.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a United States national stage filing under 35U.S.C. §371 of international application No. PCT/EP01/09251, filed Aug.10, 2001, designating the United States, and claims priority to EuropeanAppln. No. 00402393.3, filed Aug. 30, 2000.

SUMMARY OF THE INVENTION

The present invention concerns nitroderivatives of polysaccharidescomprising repeating units formed of a uronic acid and a hexosamineresidue.

STATE OF THE ART

There is a large body of evidence in literature showing that NO plays acritical role in a variety of physiological and pathological conditions,opening up the possibility of therapeutical applications. NO acts as amessenger molecule which conveys biochemical signals in differentbiological systems, such as cardiovascular, central nervous and immunesystems. However, despite its beneficial effects, high concentration ofNO, or its administration under uncontrolled conditions, can displayadverse effects (e.g., hypotension, tachyfilaxis, headache,cytotoxicity, etc.).

It is therefore highly desirable to develop new drugs that, whiledelivering controlled amounts of NO into tissues, have a better safetyprofile.

Heparins are in clinical use from many years as anti-coagulant andanti-thrombotic drugs. Despite that, current treatment of hospitalisedand home patients has a considerable risk of adverse effects includingbleeding, osteoporosis and thrombocytopenia.

Compositions comprising heparin and NO-releasing compounds are used inthe clinical practice for the treatment of cardiovascular diseases.

Furthermore, recently J. E Saavedra et al, Bioorganic & MedicinalChemistry Letters 10 (2000) 751–753, descibe heparin/diazeniumdiolatesconjugates that generate nitric oxide. However, the synthesis of thesecompounds is very complicated. In addition, the specific NO releasinggroup introduced in the heparin chain has various drawbacks and,consequently it is not suitable for pharmaceutical application.

The preparation of an alginic nitric acid ester and its use in themanufacture of celluloid is disclosed in GB 417,556.

DESCRIPTION OF THE INVENTION

The present invention provides a new class of compounds presenting ahigh compatibility with tissues and organic fluids. Such new compoundsare polysaccharides containing nitro groups covalently bonded to thesaccharide structure. More specifically, the present invention providespolysaccharides essentially formed of units of uronic acid and/orhexosamine, containing nitro groups —ONO₂ covalently bonded to thesaccharide structure. Preferably, the polysaccharides according to theinvention are prevalently formed of disaccharide repeating units formedof uronic acid and hexosamine.

In a preferred embodiment of the invention, these compounds are able torelease NO in biological fluids/tissues at levels that aretherapeutically effective. The bioactive NO could be selectivelyreleased to the biological target, avoiding the risk of systemicundesired events.

In another preferred embodiment of the invention, the compoundsaccording to the invention significantly reduce the side effecttypically associated to the therapeutic use of heparin.

In a particularly preferred embodiment, both effects are present at thesame time. This result has been achieved by functionalizingpolysaccharides essentially formed of units of uronic acid and/orhexosamine, with substituents containing a —ONO₂ group. Thesesubstituents are bonded to the saccharide structure through a covalentbond. The polysaccharides according to the invention are formed of anumber of saccharide units which preferably vary from 2 to 100 units. Itis therefore clear to the person skilled in the art, that within thedefinition of polysaccharides also disaccharides and oligosaccharidesare included. In fact, nitroderivatives of di- and oligosaccharidespresent the same advantages of nitroderivatives of polysaccharides withhigher molecular weight. In the case of disaccharides, at least one ofthe two saccharides is either a uronic acid or a hexosamine. Preferably,the disaccharides according to the invention are formed of a uronic acidunit and a hexosamine unit.

The polysaccharides according to the invention are preferably preparedfrom natural glycosaminoglycanes (GAGs) prevalently formed of the unitsdefined in Schema 1.

wherein Ch-4S represents chondroitine 4 sulfate (chondroitine C), and nusually varies from 10 to 50; Ch-6S represents chondroitine 6 sulfate(chondroitine A), and n usually varies from 10 e 50; HA representshyaluronic acid and n usually varies from 10 to 250; DeS representsdermatansulfate (chondroitine B), and n usually varies from 10 e 100; HSrepresents heparansulfate, and n usually varies from 8 to 50, and HEPrepresents heparin and n usually varies from 8 to 35.

These GAGs can be directly used as a starting product in the preparationof the nitroderivatives according to the invention or they can bechemically modified according to techniques well known in the art.

For example, N-acetyl or N-sulfate groups of the hexosamino residue canbe transformed into amino groups through desulfation or deacetylationreactions; the free amino groups can be N-acetylated or N-sulfated; thesulfate groups of uronic acids can give rise to epoxy-groups throughdesulfation reaction; free amino groups and epoxy groups can be used tofix the polysaccharide to a polymeric support, as described in WO99/27976 (Baxter).

It is also known the generation of a CH₂OH group by desulfation of theposition 6 of hexosamine; furthermore other reactive positions aregenerated through deamination and resulting depolymerization; in thisway di- and oligosaccharides are obtained that present a terminal CHOwhich, by further reaction, gives rise to a new CH₂OH or a new aminogroup:

The depolymerization reaction can also take place without modifying thehexosamine ring, for example by enzymatic hydrolysis with liase orhydrolase, by chemical depolymerization with mineral acids (e.g.sulfuric acid or hydrochloric acid) or by demolition reaction of Smith.Another possible chemical modification is the supersulfation withadducts of pyridine-sulfur trioxide (K. Nagasawa; H. Uchiyama; N.Wajima, Carbohydr. Res. 158 (1986), 183–190; A. Ogamo, A. Metori; H.Uchiyama; K. Nagasawa, Carbohydr. Res. 193 (1989), 165–172; R. N. Rey;K. G. Ludwig-Baxter; A. S. Perlin, Carbohydr. Res. 210 (1991), 299–310)or with a mixture of sulfuric and chlorosulfonic acids (Naggi e al.,Biochem. Pharmacol. 36, 1895–1900, 1987).

Natural GAGs and their derivatives obtained according to the abovedefined methods show a very high affinity, amongst others, towardsendothelium, platelets and leukocytes. Their use as a carrier of a groupcapable of releasing NO such as a nitro group allows a selective useand, consequently, a reduced dosing of the active principle. Preferredpolysaccharides are heparin, heparansulfate, chondroitine A, B and C andtheir desulfated derivatives. Most preferred is heparin.

The nitration can take place in different positions of the saccharideunits of polysaccharides of molecular weight comprised between about 500(disaccharide) and about 30.000 (high molecular weight polysaccharide).The preferred positions for the introduction of the nitro-containinggroups are the following: CH₂OH in 6 of hexosamine, CHOH in 3 ofhexosamine and of uronic acid, carboxy group in 6 of uronic acid, CHOHin 2 of (desulfated) uronic acid and NH₂ of desulfated or deacetylatedhexosamine.

The nitro group can be covalently bonded to the saccharide structureeither directly (e.g. through nitration of a carbon of the saccharideunit) or through a divalent radical acting as a spacer between thepolysaccharide unit and the nitro group. Suitable spacers are divalentradicals derived from C₂–C₂₀ aliphatic or aromatic hydrocarbons, ethers,polyethers, carboxylic acids or their derivatives, and the like.

In the following we will refer to the nitroderivatives of heparin, butit is clear to the skilled person that, by analogy, it is possible toprepare the nitroderivatives of any other polysaccharide essentiallyformed of units of uronic acid and/or hexosamine such as GAGs or theirderivatives as described above.

The nitroderivatives according to the invention can optionally be usedin combination with a compound that donates, transfers or releasesnitric oxide as a charges specie, i.e. nitrosonium (NO⁺) or nitroxyl(NO⁻), or as the neutral specie, nitric oxide (NO⁻) and/or a compoundthat stimulates endogenous production of NO or EDRF (Endothelium DerivedRelaxing Farctor) in vivo.

A first path to the preparation of nitroderivatives according to theinvention is by direct nitration by means of a nitrating mixture. Thereaction can be performed on different substrates. For example, it ispossible to use heparin sodium salt, 6-O-desulfated heparin andN-deacetylated heparin. The reaction is preferably carried out by firstpreparing the nitrating mixture, to whom the previously dried heparin isadded in small amounts under stirring. Examples of nitrating mixturesare: sulfuric acid-nitric acid, phosphoric acid-nitric acid, aceticanhydride-nitric acid, nitrous oxide-sulfuric acid. Preferred mixtureare the mixture sulfuric acid-nitric acid, also called sulfo-nitricmixture and the mixture acetic anhydride-nitric acid.

When willing to limit possible depolymerization reactions, the reactionis preferably thermostated at low temperature, e.g. by using a ice-bathor refrigerating mixtures. In case of sulfo-nitric mixture, the molarratio between sulfuric acid and nitric acid can vary in a broad rangeand is preferably comprised between 5:1 and 1:2, more preferably between3:1 and 1:1.25. The molecular weight of the nitroheparin can varyaccording to the used reaction conditions and is preferably comprisedbetween 500 and 20.000, most preferably between 500 and 12.000. Theamount of nitro groups per saccharide unit also varies considerably, andthe ratio in equivalents between saccharide units and nitro groups ispreferably comprised between 40:1 and 2:3, more preferably comprisedbetween 20:1 and 2:3, most preferably between 10:1 and 1:1.

Another synthetic path for the synthesis of nitroderivatives ofpolysaccharides according to the invention is by haloacylation followedby nitration. In this case it is possible to prepare first apolysaccharide containing N-desulfated hexosamine. The N-desulfatedhexosamine is then reacted with a halo anhydride such as iodoaceticanhydride, obtaining the corresponding amide in position 2 of thehexosamine. The iodine atom is then substituted with a nitro group byreaction with a suitable nitrate. By using the suitable reactionconditions it is possible to dose the amount of N-desulfation and,consequently, the amount of nitro groups introduced. Examples ofsuitable nitrates are AgNO₃ and NBu₄NO₃. It is possible to useanhydrides different from iodoacetic anhydride, such as the anhydride ofa ω-halocarboxylic acid, obtaining in this way nitroderivatives having adifferent spacer group. Preferred halogens are iodine and bromine. Morepreferred is iodine. In the case of heparin and iodoacetic anhydride asstarting materials, the reaction scheme is the following:

It is also possible to acylate the position 6 of desulfated hexosamine.In this case it is preferable to use compounds of formulaX—(CH₂)_(n)—COY wherein X is chlorine, bromine, iodine or tosyl;preferably X is bromine; Y is chlorine, mercaptothiazole,mercaptobenzothiazole, mercaptobenzoxazole, p-nitrophenol. The reactionscheme for heparin is the following:

Experimental Section

Heparin sodium salt from pig intestinal mucosa (HEP) was bought fromLaboratorio Derivati Organici. The analytical characteristics were thefollowing:

Elemental analysis: C: 21.26%; N: 2.07%;

-   -   atom of N per saccharide: 0.5        Synthesis 1        Preparation of 50% N-desulfated Heparin (HEP 1)

An excess of pyridine was added to an aqueous solution of 2 g of HEP,previously eluted from a column of Amberlite IR 120 (H⁺). The solutionwas evaporated under reduced pressure; the resulting pyridine salt ofthe heparin was dissolved in 100 ml of a mixture of DMSO/methanol 95:5and stirred at 20° C. for 2 hours, in order to obtain a desulfationdegree of about 50%.

Then, the solution was diluted with an equal volume of distilled water.The pH was adjusted to about 10 by addition of sodium hydroxide 1M andthe solution was dialyzed against distilled water in membranes (cut-off1000–2000 D). The final product was isolated by evaporation underreduced pressure.

UV: [C]=0.73 mg/ml; abs=1.469; λ=195 nm

Synthesis 2

Preparation of 100% N-desulfated Heparin (HEP 2)

Synthesis 1 was repeated, reacting HEP for 8 h instead of 2 h. 100%N-desulfated heparin was obtained.

UV: [C]=0.11 mg/ml; abs=0.355; λ=195 nm

Synthesis 3

Preparation of N- and 100% 6-O-desulfated Heparin (HEP 3)

An excess of pyridine was added to an aqueous solution of 5 g of HEP,previously eluted from a column of Amberlite IR 120 (H⁺). The solutionwas evaporated under reduced pressure; the resulting pyridine salt ofthe heparin was dissolved in 500 ml of a mixture of DMSO/methanol 90:10and stirred at 65° C. for 8 hours. Then, the solution was diluted withan equal volume of distilled water, neutralized by addition of sodiumhydroxide 1M and purified via ultrafiltration. The final product wasisolated by evaporation under reduced pressure.

Synthesis 4

Preparation of 6-O-desulfated Heparin (HEP 4)

6-O-desulfated heparin was prepared via N-resulfation of N- andO-desulfated heparin (HEP3)

9.6 g of sulfur trioxide trimethylamine complex (TMA-SO₃) were added toan aqueous solution of 4 g of HEP 3, previously saturated with sodiumhydrogen carbonate and thermostated at 55° C. After 20 hours 9.6 g ofTMA-SO₃ were added and the mixture stirred at 55° C. for 7 hours. Then,the mixture was poured into ethanol and, after 15–20 hours at 4° C.,filtered on frittered glass filter. The precipitate was dissolved inwater and dialyzed against distilled water in membranes (cut-off2000–1000 D). The aqueous solution was evaporated under reducedpressure.

The product has the following characteristics:

UV: [C]=1.1 mg/ml; abs=1.976; λ=195 nm

-   -   0.11 mg/ml; abs=0.206; λ=195 nm        Synthesis 5        Preparation of N-acetylated Heparin (HEP 5)

N-acetylated heparin was prepared by N-acetylation of 100% N-desulfatedheparin (HEP 2).

600 mg of HEP 2 were dissolved in 6 ml of distilled water; the solutionwas cooled to 0° C. and saturated with sodium hydrogen carbonate; 500 μlof acetic anhydride were added to this solution and the mixture wasstirred for 2 hours at 0° C. During the reaction, pH was controlled andmaintained at about 8 by adding sodium hydrogen carbonate. Then, thesolution obtained was dialyzed against distilled water in membranes(cut-off 2000–1000 D).

The aqueous solution was evaporated under reduced pressure.

Synthesis 6

Preparation of 100% N-desulfated Supersulfated Heparin (HEP 6)

Synthesis 2 was repeated using as a starting material supersulfatedheparin prepared according to the procedure described in Naggi e al.,Biochem. Pharmacol. 36, 1895–1900, 1987. The product obtained wasacetylated as described in synthesis 5.

Elemental analysis: C: 15.34%; N: 1.28%;

-   -   atom of N per saccharide: 0.5

UV: [C]=0.11 mg/ml; abs=0.830; λ=195 nm

Conductimetric titration: SO₃ ⁻/COO⁻=3.7

Synthesis 7

Preparation of 100% N-acetylated Desulfated Heparin (HEP 7)

N-acetylated almost completely O-desulfated heparin was prepared byacetylation of almost completely N,O-desulfated heparin.

An excess of pyridine was added to an aqueous solution of 3 g of HEP,previously eluted from a column of Amberlite IR 120 (H⁺). The solutionwas evaporated under reduced pressure; the resulting pyridine salt ofthe heparin was dissolved in 150 ml of a mixture of DMSO/methanol 90:10and stirred at 100° C. for 24 hours, in order to obtain totallyN-desulfation and almost completely O-desulfation.

Then, the solution was diluted with an equal volume of distilled water.The pH was adjusted to about 9 by addition of sodium hydroxide 1M andthe solution was dialyzed against distilled water in membranes (cut-off1000–2000 D). The final product was isolated by evaporation underreduced pressure.

The product obtained was acetylated as described in synthesis 5.

UV: [C]=0.11 mg/ml; abs=1.660; λ=195 nm

Conductimetric titration: SO₃ ⁻/COO⁻=1.1

Synthesis 8

Preparation of 100% N-acetylated 50% 6-O-desulfated Heparin (HEP 8)

This heparin derivative was prepared by acetylation of 50%6-O-desulfated heparin., according with procedure described in synthesis3, reacting HEP for 6 h instead of 8.

UV: [C]=0.11 mg/ml; abs=1.356; λ=195 nm

Conductimetric titration: SO₃ ⁻/COO⁻=1.1

Example 1

Preparation of Nitroheparin via Nitration of Heparin (HEP) withSulfo-nitric Mixture

Sulfo-nitric mixture was prepared by dropping 10 ml of nitric acid (90%)in 20 ml of sulfuric acid (96%) kept under stirring and cooled at 0° C.with an ice bath. When no more smoke was observed, 1 g of HEP was addedin portions over an hour. The mixture was then stirred for 1 h at roomtemperature.

At the end, the mixture was poured into 500 ml of diethyl ether cooledin a bath of acetone/CO₂. The cool mixture was filtered on a fritteredglass filter under reduced pressure. A sticky solid remained on thefilter, that was washed with cool ether and then recovered from thefilter by washing it with an aqueous solution of sodium hydrogencarbonate. The solution obtained, at pH 8, was concentrated underreduced pressure and dialyzed against distilled water in membranes at1000 D.

The aqueous solution is evaporated at room temperature under reducedpressure.

The product has the following characteristics:

UV: [C]=0.11 mg/ml; abs=1.513; λ=195 nm

Conductimetric titration: SO₃ ⁻/COO⁻=3.8

Example 2

Preparation of Nitroheparin via Nitration of Heparin (HEP) withSulfo-nitric Mixture

Sulfo-nitric mixture was prepared by dropping 10 ml of nitric acid (90%)in 20 ml of sulfuric acid (96%) kept under stirring and cooled at 0° C.with an ice bath. When no more smoke was observed, 1 g of HEP was addedin portions over an hour. The mixture was then stirred for 1 h at 0° C.

At the end, the mixture was poured into 300 ml of diethyl ether cooledin a bath of acetone/CO₂. The cool mixture was filtered on a fritteredglass filter under reduced pressure. A sticky solid remained on thefilter, that was washed with cool ether and then recovered from thefilter by washing it with an aqueous solution of sodium hydrogencarbonate. The solution obtained, at pH 8, was concentrated underreduced pressure and dialyzed against distilled water in membranes at1000 D.

The aqueous solution is evaporated at room temperature under reducedpressure.

The product has the following characteristics:

Elemental analysis: C: 17.10%; N: 4.01%;

-   -   atom of N per saccharide: 1.2    -   ONO₂ per saccharide: 0.7

UV: [C]=0.11 mg/ml; abs=1.586; λ=195 nm

Conductimetric titration: SO₃ ⁻/COO⁻=3.5

MW: 9150

Example 3

Preparation of Nitroheparin via Nitration of Heparin (HEP) withSulfo-nitric Mixture

Sulfo-nitric mixture was prepared by dropping 16.3 ml of nitric acid(90%) in 13.8 ml of sulfuric acid (98%) kept under stirring, and cooledat 0° C. in an ice bath. When no more smoke was observed, 1 g of HEP wasadded in portions over an hour. The mixture was then stirred for 1 h atroom temperature

At the end, the mixture was poured into 500 ml of diethyl ether cooledin a bath of acetone/N₂. The cool mixture was filtered on a fritteredglass filter under reduced pressure. A sticky solid remained on thefilter, that was washed with cool ether and then recovered from thefilter by washing it with an aqueous solution of sodium hydrogencarbonate. The solution obtained, at pH 8, was concentrated underreduced pressure and dialyzed against distilled water in membranes at1000 D.

The aqueous solution was evaporated at room temperature under reducedpressure.

The product has the following characteristics:

UV: [C]=0.073 mg/ml; abs=1.358; λ=195 nm

MW: 8000

Example 4

Preparation of Nitroheparin via Nitration of 6-O-desulfated Heparin (HEP4) with Sulfo-nitric Mixture

Sulfo-nitric mixture was prepared by dropping 10 ml of nitric acid (90%)in 20 ml of sulfuric acid (98%) kept under stirring and cooled at 0° C.with an ice bath. When no more smoke was observed, 1 g of HEP 4 wasadded in portions over an hour. The mixture was then stirred for 1 h atroom temperature

At the end, the mixture was poured into 500 ml of diethyl ether cooledin a bath of acetone/N₂. The cool mixture was filtered on a fritteredglass filter under reduced pressure. A sticky solid remained on thefilter, that was washed with cool ether and then recovered from thefilter by washing it with an aqueous solution of sodium hydrogencarbonate. The solution obtained, at pH 8, was concentrated underreduced pressure and dialyzed against distilled water in membranes(cut-off 2000–1000 D).

The aqueous solution was evaporated at room temperature under reducedpressure.

The product has the following characteristics:

Elemental analysis: C: 20.61%; N: 3.86%;

-   -   atom of N per saccharide: 1    -   ONO₂ per saccharide: 0.5

UV: [C]=0.11 mg/ml; abs=1.176; λ=195 nm

Conductimetric titration: SO₃ ⁻/COO⁻=3.0

MW: 22400

Example 5

Preparation of Nitro Heparin via Nitration of 6-O-desulfated Heparin(HEP 4) with Sulfo-nitric Mixture

Sulfo-nitric mixture was prepared by dropping 16.3 ml of nitric acid(90%) in 13.8 ml of sulfuric acid (98%) kept under stirring and cooledat 0° C. with an ice bath. When no more smoke was observed, 1 g of HEP 4was added in portions over an hour. The mixture was then stirred for 95′at room temperature

At the end, the mixture was poured into 300 ml of diethyl ether cooledin a bath of acetone/N₂. The cool mixture was filtered on a fritteredglass filter under reduced pressure. A sticky solid remained on thefilter, that was washed with cool ether and then recovered from thefilter by washing it with an aqueous solution of sodium hydrogencarbonate. The solution obtained, at pH 8, was concentrated underreduced pressure and dialyzed against distilled water in membranes at2000 D.

The aqueous solution was evaporated at room temperature under reducedpressure.

The product has the following characteristics:

Elemental analysis: C: 14.18%; N: 3.42%;

-   -   atom of N per saccharide: 1.25    -   ONO₂ per saccharide: 0.75

UV: [C]=0.11 mg/ml; abs=1.489; λ=195 nm

Example 6

Preparation of Nitroheparin via Nitration of N-acetylated Heparin (HEP5) with Sulfo-nitric Mixture

Sulfonitric mixture was prepared by dropping 1.32 ml of nitric acid(90%) in 2.64 ml of sulfuric acid (98%) kept under stirring and cooledat 0° C. in an ice bath. When no more smoke was observed, 132 mg of HEP5 were added in portions over an hour. The mixture was then stirred 2hours at 0° C. and, after cooling in an acetone/N₂ bath, poured into anaqueous solution saturated with sodium hydrogen carbonate. Other basicsolution was added until pH 7 was reached, then the solution was frozenand lyophilized. The solid was then dialyzed against distilled water inmembranes (cut-off 2000–1000 D).

The aqueous solution was evaporated at room temperature under reducedpressure.

The product has the following characteristics:

Elemental analysis: C: 21.89%; N: 4.95%;

-   -   N per saccharide: 1.15    -   ONO₂ per saccharide: 0.65

UV: [C]=0.05 mg/ml; abs=1.574; λ=195 nm

MW: 15700

Example 7

Preparation of Nitroheparin via Nitration of N-acetylated DesulphatedHeparin (HEP 7) with Sulfo-nitric Mixture

Sulfonitric mixture was prepared by dropping 2.4 ml of nitric acid (90%)in 4.8 ml of sulfuric acid (96%) kept under stirring and cooled at 0° C.in an ice bath. When no more smoke was observed, 240 mg of HEP 7 wereadded in portions over 2 hour and 15 minutes. The mixture was thenstirred an hour at 0° C. and 10 minutes at r.t. After cooling in anacetone/N₂ bath, it was poured into an aqueous solution saturated withsodium hydrogen carbonate. Other basic solution was added until pH 7 wasreached, then the solution was frozen and lyophilized. The solid wasthen dialyzed against distilled water in membranes (cut-off 1000 D).

The aqueous solution was evaporated at room temperature under reducedpressure.

The product has the following characteristics:

Elemental analysis: C: 25.90%; N: 6.27%;

-   -   N per saccharide: 1.45    -   ONO₂ per saccharide: 0.45

UV: [C]0.11 mg/ml; abs=1.960; λ=195 nm

Conductimetric titration: SO₃ ⁻/COO⁻=1.1

MW: 13500

Example 8

Preparation of Nitroheparin via Nitration of N-acetylated 50%6-0-Heparin (HEP 8) with Sulfo-nitric Mixture

Sulfonitric mixture was prepared by dropping 5 ml of nitric acid (90%)in 10 ml of sulfuric acid (96%) kept under stirring and cooled at 0° C.in an ice bath. When no more smoke was observed, 500 mg of HEP 8 wereadded in portions over an hour. The mixture was then stirred an hour at0° C. and 10 minutes at r.t. After cooling in an acetone/N₂ bath, themixture was poured into an aqueous solution saturated with sodiumhydrogen carbonate. Other basic solution was added until pH 7 wasreached, then the solution was frozen and lyophilized. The solid wasthen dialyzed against distilled water in membranes (cut-off 1000 D).

The aqueous solution was evaporated at room temperature under reducedpressure. The solid obtained is purified via gel-chromatography.

The product has the following characteristics:

Elemental analysis: C: 17.16%; N: 3.42%;

-   -   N per saccharide: 1.2    -   ONO₂ per saccharide: 0.2

UV: [C]=0.11 mg/ml; abs=1.429; λ=195 nm

Conductimetric titration: SO₃ ⁻/COO⁻=1.1

MW: 12100

Example 9

Preparation of Nitroheparin via Nitration of N-acetylated Heparin (HEP5) with Sulfo-nitric Mixture

Sulfonitric mixture was prepared by dropping 4.89 ml of nitric acid(90%) in 4.23 ml of sulfuric acid (96%) kept under stirring and cooledat 0° C. in an ice bath. When no more smoke was observed, 300 mg of HEP5 were added in portions over an hour. The mixture was then stirred another hour at 0° C. and, after cooling in an acetone/N₂ bath, pouredinto an aqueous solution saturated with sodium hydrogen carbonate. Otherbasic solution was added until pH 7 was reached, then the solution wasfrozen and lyophilized. The solid was then dialyzed against distilledwater in membranes (cut-off 1000 D).

The aqueous solution was evaporated at room temperature under reducedpressure.

The product has the following characteristics:

Elemental analysis: C: 22.58%; N: 4.91%;

-   -   N per saccharide: 1.2    -   ONO₂ per saccharide: 0.2

Conductimetric titration: SO₃ ⁻/COO⁻=1.9

Example 10

Preparation of Nitroheparin via Nitration of N-acetylated Heparin (HEP5) with Nitro-acetic Mixture

Nitroacetic mixture was prepared by dropping, over 45 minutes, 18.5 mlof acetic anhydride in 28 ml of nitric acid (90%) kept under stirringand cooled at −20° C. in an N₂/acetone bath. At the end, 250 mg of HEP 5were added. When the temperature was stabilized, N₂/acetone bath waschanged with an ice bath; the mixture was stirred 3.5 hours at 0° C. and30 minutes at r.t. The mixture was poured into 700 ml of cold distilledwater and neutralized with NaOH 1M. The resulting aqueous solution wastreated with bioconcentrator Mini-Plate (10000 D) and lyophilized.

The product has the following characteristics:

Elemental analysis: C: 23.74%; N: 5.01%;

-   -   N per saccharide: 1.25    -   ONO₂ per saccharide; 0.75

Conductimetric titration: SO₃ ⁻/COO⁻=1.1

Example 11

Preparation of Nitroheparin via N-iodoacylation and Nitration of 50%N-desulfated Heparin (HEP 1)

386 mg of diiodoacetic anhydride were added to an aqueous solution of200 mg of HEP 1, cooled at 0° C. and saturated with sodium hydrogencarbonate. The mixture was stirred for 2 hours at 0° C. and then left at4° C. for about 80 hours. Then, the obtained solution was dialyzedagainst distilled water in membranes (cut-off 2000–1000 D). 255 mg ofcalcium nitrate and 612 mg of silver nitrate were added to this solutionat 4° C. The mixture was stirred in this conditions for 48 hours andthen filtered on a frittered glass filter under reduced pressure. Theexcess of silver nitrate was eliminated by precipitating silver chlorideby addition of sodium chloride. The solution was poured into ethanol andcooled at 4° C. for about 70 hours. The precipitate was filtered andwashed with acetone and diethyl ether.

The product has the following characteristics:

UV: [C]=0.073 mg/ml; abs=0.954; λ=195 nm

Elemental analysis: C: 19.63%; N: 1.83%;

-   -   N per saccharide: 0.56    -   ONO₂ per saccharide: 0.06

Conductimetric titration: SO₃ ⁻/COO⁻=1.1

Example 12

Preparation of Nitroheparin via N-iodoacetylation and Nitration of 100%N-desulfated Heparin (HEP 2).

193 mg of diiodoacetic anhydride were added to 2 ml of an aqueoussolution of 100 mg of HEP 2, cooled at 4° C. and saturated with sodiumhydrogen carbonate for about 48 h. The solution freeze dried. Theobtained solid was suspended in 6 ml of water and neutralized. 128 mg ofcalcium nitrate and 306 mg of silver nitrate were added to this solutionat 4° C. The mixture was stirred in this conditions for 24 h andfiltered on a frittered glass filter under reduced pressure. The excessof silver nitrate eliminated precipitating silver chloride by additionof sodium chloride. The solution was poured into ethanol and cooled at4° C. for about 18 h. The precipitate was filtered, solubilized withwater and the solution freeze dried.

The product was analyzed by ¹³C NMR spectroscopy. The presence of CH₃Igroup is pointed out by signal at 1.2 ppm. The sample, solubilized in 4ml of water, was further treated with 128 mg of calcium nitrate and 306mg of silver nitrate at 4° C. The mixture was stirred in this conditionsfor 48 h and filtered on a frittered glass filter under reducedpressure. The excess of silver nitrate eliminated precipitating silverchloride by addition of sodium chloride. The solution was poured intoethanol and cooled at 4° C. for about 18 h. The precipitate wasfiltered, solubilized with water and the solution freeze dried.

The product has the following characteristics:

UV: [C]=0.1 mg/ml; abs=1.062; λ=195 nm

Elemental analysis: C: 20.22%; N: 1.89%;

-   -   N per saccharide: 0.56    -   ONO₂ per saccharide: 0.06

Conductimetric titration: SO₃ ⁻/COO⁻=1.1

Example 13

Preparation of Nitroheparin via N-iodoacetylation and Nitration of 50%N-desulfated Heparin (HEP 1).

386 mg of diiodoacetic anhydride were added to 3 ml of an aqueoussolution of 200 mg of HEP 1, cooled at 4° C. and saturated with sodiumhydrogen carbonate for about 72 h. The solution was poured into ethanol(15 ml) and cooled at 4° C. The precipitate was filtered, solubilizedwith water and the solution freeze dried.

The obtained solid was suspended in 6 ml of water and neutralized. 255mg of calcium nitrate and 612 mg of silver nitrate were added to thissolution at 4° C. The mixture was stirred in this conditions for 48 hand filtered on a frittered glass filter under reduced pressure. Theexcess of silver nitrate eliminated precipitating silver chloride byaddition of sodium chloride. The solution was poured into ethanol andcooled at 4° C. for about 18 h. The precipitate was filtered,solubilized with water and the solution freeze dried.

The product was analyzed by ¹³C NMR spectroscopy.

The product has the following characteristics:

UV: [C]=0,1 mg/ml; abs=1,092; λ=195 nm

Elemental analysis: C: 20,25%; N: 2,10%;

-   -   N per saccharide: 0.62    -   ONO₂ per saccharide: 0.12

Conductimetric titration: SO₃ ⁻/COO⁻=1.2

Example 14

Preparation of Nitroheparin via N-iodoacetylation and Nitration ofN-desulfated Supersulfated Heparin (HEP 6).

276 mg of diiodoacetic anhydride were added to 3 ml of an aqueoussolution of 200 mg of HEP 6, cooled at 4° C. and saturated with sodiumhydrogen carbonate for about 48 h. The solution was dialyzed againstdistilled water in membranes at 1000 D, and the aqueous solution wasfreeze dried.

The obtained solid was suspended in 6 ml of water. 163 mg of calciumnitrate and 391 mg of silver nitrate were added to this solution at 4°C. The mixture was stirred in this conditions for 24 h and filtered on afrittered glass filter under reduced pressure. The excess of silvernitrate eliminated precipitating silver chloride by addition of sodiumchloride. The solution was poured into ethanol and cooled at 4° C. forabout 18 h. The precipitate was filtered, solubilized with water and thesolution freeze dried.

The product has the following characteristics:

UV: [C]=0,1 mg/ml; abs=0,913; λ=195 nm

Elemental analysis: C: 17,10%; N: 1,53%;

-   -   N per saccharide: 0.55    -   ONO₂ per saccharide: 0.05

Conductimetric titration: SO₃ ⁻/COO⁻=1.1

Pharmacological Studies

Nitro-heparins were compared with standard heparin (Liquemin-Roche).

Coagulation Assays

50 μl of test heparin solution were added to 450 μl of a pool (at least10) of healthy donor plasma. Activated partial thromboplastin time(aPTT) and thrombin clotting time (TcT) were measured by standard assayswith an automated coagulometer (ACL 300 R, Istrumentation Laboratory,Milan) as described in Momi S et al., Haematologica 2001; 86: 297–302.

Maximal acquisition time for aPTT was set at 249 s, and at 167 s forTcT.

Whole Blood Aggregation Assays

Platelet aggregation was studied in whole blood using the impedancemethod described in Gresele P et al. Thromb Haemost 1986; 55: 12–18,with a Chrono-Log whole blood aggregometer (mod. 540, Chrono-Log Corp.,Havertown, Pa., USA). Samples (1 ml) of citrated whole blood were usedto measure the change of electrical impedance between two fineelectrodes dipped in the blood under continuous stirring and afterstimulation with a platelet agonist. Initially, a monolayer of plateletscoats the electrodes. The presence of an aggregatory agent causesadditional platelets to clump and adhere to the mono layer and increasesthe impedance between the electrodes. The change in impedance isrecorded and correlated to the aggregation.

10 μl of test heparin solution or the vehicle (water) were added to 1 mlof citrated blood diluted 1:2 with citrated physiological solution andlet incubate for 2 min at 37° C. The aggregating agent ADP (2 to 10 μMto obtain a sub maximal effect) has been then added and the aggregationcurve recorded for 10 min. The maximal amplitude obtained was measuredand increases in maximal amplitude of aggregation after pre-incubationwith heparins were taken as an index of proaggregatory activity of thetest compounds.

Aggregation Studies on Platelet Rich Plasma (PRP)

Blood was collected into 1/10 v:v trisodium citrate (3.8%) from healthydonors. The blood was centrifuged at room temperature at 180×g for 15min. Platelet rich plasma was separated and the platelet count in PRPwas adjusted to 2.5×10⁸/ml with autologous platelet poor plasma (PPP).

PRP was incubated with nitro heparins for 2 or 10 min at 37° C. beforestimulation with ADP (sub threshold or threshold doses) or thresholdaggregatory concentrations of the stable thromboxane analogue U46619. Athreshold dose of an agonist was defined as the minimal amount of theinducer (as identified from dose-response curves) giving a 60% ofmaximal aggregation within 3 min. We used sub threshold doses to prove apro-aggregatory effect of the test compound, while threshold doses wereused in experiments aimed to identify an inhibitory effect of the testcompound (Vezza R, et al. Blood 1993; 82: 2704–2713).

Aggregation Studies on Gel Filtered Platelets

In experiments in which gel filtered platelets were used, 5 ml of PRP isallowed to flow through a column (Sepharose 2B): platelets are eluted inabout seven fractions, which can be identified by their turbidity. Theplatelet count was adjusted to 10⁸/ml³.

Experiments on the Role of Nitric Oxide (NO)

For experiments aimed to identify the role of NO in the inhibitoryaction on platelets, gel filtered human platelets were pre incubated for2 min with the test compound and then stimulated with U46619.

Activated Partial Thromboplastin Time (aPTT) and Thrombin clotting Time(TcT) in Vehicle, Standard Heparin (Liquemin®), and NO-Heparins TreatedHuman Plasma

TABLE 1 Compound n aPTT TcT Vehicle 9 24.5 ± 1.1   16 ± 0.4 Liquemin ® 9163.5 ± 19.8 167 ± 0  Ex. 1 3 24.4 ± 1.7 15.9 ± 0.4 Ex. 3 3 24.5 ± 2  16.3 ± 0.5 Ex. 4 3 57.8 ± 1.3 167 ± 0  Ex. 5 3 29.2 ± 0.3 18.9 ± 0.4 Ex.7 3 25.8 ± 2.5 19.2 ± 0.8 Ex. 9 4 30.5 ± 2.3   37 ± 5.9 Values areexpressed as mean ± standard error mean (s.e.m.).Effect of Vehicle, Standard Heparin (Liquemin®), and NO-Heparins on ADPand U46619 Induced Human Platelet Aggregation in PRP (Platelet RichPlasma)

TABLE 2 ADP U46619 Compound n % potentiation % inhibition Vehicle 5 0  0Liquemin ® 4 87.7 −9 Ex. 1 4 −12.8 62 Ex. 3 4 −12.8 50 Ex. 4 4 52 n.a.Ex. 5 3 −12.7 28 Ex. 7 3 −29 n.a. Ex. 9 3 −9.2 n.a. PRP aggregation wasobtained by addition of sub-threshold doses of the aggregating agentADP, or of threshold doses of the thromboxane stable analogue U46619.Values are expressed as percent of control (vehicle) aggregation asmean. n.a.: not assessed

1. Polysaccharides essentially formed of units of uronic acid andhexosamine, wherein said uronic acid and/or said hexosamine units aresulfated, said polysaccharide containing —ONO₂ groups covalently bondedto the saccharide structure.
 2. Polysaccharides according to claim 1wherein the polysaccharides are prevalently formed of disacchariderepeating units formed of uronic acid and hexosamine.
 3. Polysaccharidesaccording to claim 1 wherein the —ONO₂ group is directly attached tosaid polysaccharide.
 4. Polysaccharides according to claim wherein the—ONO₂ group is covalently bonded to the saccharide structure through adivalent radical R acting as a spacer between the polymeric chain andthe —ONO₂ group, wherein R is a divalent C₂–C₂₀ aliphatic or aromatichydrocarbon radical.
 5. Polysaccharides according to claim 1 wherein thenumber of saccharide units varies between 2 and
 100. 6. Polysaccharidesaccording to claim 5 wherein the number of saccharide units variesbetween 4 and
 70. 7. Disaccharides according to claim 1 formed of auronic acid and a hexosamine unit.
 8. Polysaccharides according to claim1 wherein the equivalents ratio between saccharide units and —ONO₂groups varies from 20:1 to 2:3.
 9. Polysaccharides according to claim 8wherein the equivalents ratio between saccharide units and —ONO₂ groupsvaries from 10:1 to 1:1.
 10. Polysaccharides according to claim 1,wherein the —ONO₂ groups are introduced by reaction of one of thefollowing positions: CH₂OH in position 6 of hexosamine, CHOH in position3 of hexosamine and of uronic acid, carboxy group in position 6 ofuronic acid, CHOH in position 2 of (desulfated) uronic acid, and NH₂ ofdesulfated or deacetylated hexosamine.
 11. Polysaccharides according toclaim 1 comprised of polymerized glycosaminoglycans selected from thegroup consisting of chondroitin sulfate, dermatansulfate,heparansulfate, and heparin.
 12. Polysaccharides according to claim 11,wherein said chondroitin sulfate is selected from the group consistingof chondroitin 4 sulfate chondroitin C) and chondroitin 6 sulfatechondroitin A).
 13. Polysaccharides according to claim 11, wherein saidglycosaminoglycans are selected from the group consisting ofheparansulfate and heparin.
 14. A process for the preparation ofnitroderivatives of polysaccharides according to claim 1, comprisingintroducing the —ONO₂ groups by reaction of the polysaccharide with anitrating mixture at one of the following positions: CH₂OH in position 6of hexosamine, CHOH in position 3 of hexosamine and of uronic acid,carboxy group in position 6 of uronic acid, CHOH in position 2 of(desulfated) uronic acid, and NH₂ of desulfated or deacetylatedhexosamine.
 15. The process according to claim 14 wherein the nitratingmixture is selected from the group consisting of: sulphuric acid-nitricacid and acetic anhydride-nitric acid.
 16. The process according toclaim 15 wherein the polysaccharide is selected from the groupconsisting of heparin and depolymerized heparin.
 17. The processaccording to claim 14, wherein the polysaccharides are selected from thegroup consisting of chrondroitin sulfate, dermatansulfate,heparansulfate, and heparin.
 18. The process according to claim 17,wherein said chondroitin sulfate is selected from the group consistingof chondroitin 4 sulfate (chondroitin C) and chondroitin 6 sulfate(chondroitin A).
 19. The process according to claim 17, wherein saidpolysaccharides are selected from the group consisting of heparansulfateand heparin.
 20. Heparin and depolymerized heparin, with a molecularweight comprised between 500 and 21,000 Daltons, containing —ONO₂ groupscovalently bonded to the saccharide structure.
 21. Heparin anddepolymerized heparin according to claim 20 wherein the equivalentsratio between saccharide units and —ONO₂ groups varies from 20:1 to 2:3.22. Heparin and depolymerized heparin according to claim 21 wherein theequivalents ratio between saccharide units and —ONO₂ groups varies from10:1 to 1:1.
 23. Heparin and depolymerized heparin according to claim 20wherein the —ONO₂ group is directly attach to said heparin anddepolymerized heparin.
 24. Heparin and depolymerized heparin accordingto claim 20 wherein the —ONO₂ group is covalently bonded to thesaccharide structure through a divalent radical R acting as a spacerbetween the polymeric chain and the —ONO₂ group, wherein R is a divalentC₂–C₂₀ aliphatic or aromatic hydrocarbon radical.
 25. A method forinhibiting coagulation in an individual in need thereof comprisingadministering to said individual a heparin or depolymerized heparin asrecited in any one of claims 20, 21, or
 22. 26. A method for inhibitingthrombosis in an individual in need thereof comprising administering tosaid individual a heparin or depolymerized heparin according to any oneof claims 20, 21, or
 22. 27. A pharmaceutical composition comprising aheparin or depolymerized heparin according to any one of claims 20, 21,or 22 and a pharmaceutically acceptable carrier.